Synthesis and SAR of Tetracyclic Inhibitors of Protein Kinase CK2 Derived from Furocarbazole W16

Abstract The serine/threonine kinase CK2 modulates the activity of more than 300 proteins and thus plays a crucial role in various physiological and pathophysiological processes including neurodegenerative disorders of the central nervous system and cancer. The enzymatic activity of CK2 is controlled by the equilibrium between the heterotetrameric holoenzyme CK2α2β2 and its monomeric subunits CK2α and CK2β. A series of analogues of W16 ((3aR,4S,10S,10aS)‐4‐{[(S)‐4‐benzyl‐2‐oxo‐1,3‐oxazolidin‐3‐yl]carbonyl}‐10‐(3,4,5‐trimethoxyphenyl)‐4,5,10,10a‐tetrahydrofuro[3,4‐b]carbazole‐1,3(3aH)‐dione ((+)‐3  a)) was prepared in an one‐pot, three‐component Levy reaction. The stereochemistry of the tetracyclic compounds was analyzed. Additionally, the chemically labile anhydride structure of the furocarbazoles 3 was replaced by a more stable imide (9) and N‐methylimide (10) substructure. The enantiomer (−)‐3  a (K i=4.9 μM) of the lead compound (+)‐3  a (K i=31 μM) showed a more than sixfold increased inhibition of the CK2α/CK2β interaction (protein‐protein interaction inhibition, PPII) in a microscale thermophoresis (MST) assay. However, (−)‐3  a did not show an increased enzyme inhibition of the CK2α2β2 holoenzyme, the CK2α subunit or the mutated CK2α′ C336S subunit in the capillary electrophoresis assay. In the pyrrolocarbazole series, the imide (−)‐9  a (K i=3.6 μM) and the N‐methylimide (+)‐10  a (K i=2.8 μM) represent the most promising inhibitors of the CK2α/CK2β interaction. However, neither compound could inhibit enzymatic activity. Unexpectedly, the racemic tetracyclic pyrrolocarbazole (±)‐12, with a carboxy moiety in the 4‐position, displays the highest CK2α/CK2β interaction inhibition (K i=1.8 μM) of this series of compounds.


Summary of the specific optical rotation
Calculation: manual integration. Calculation method: area %, use of blank subtraction from the same series.

Synthesis of (S)-4 and (R)-4
Procedures for the synthesis of 15, 11, and (S)-4 are already described in literature.
Here, modified syntheses and additional spectroscopic data are given.
The layers were separated and the aqueous layer was extracted with CH2Cl2 (3 x 75 mL). The combined organic layers were dried (Na2SO4), filtered and concentrated in vacuo. The residue was suspended in EtOH (50 mL) and the mixture was put into an ultrasonic bath for 30 min. The slurry was cooled down (-20 °C), filtered and the solid was washed with cold EtOH (3 x 30 mL) and cold Et2O (4 x 30 mL).
Exceptions and special features: For compound (+)-3d one phenyl group and one methoxy group and for compound (+)-14d three methoxy groups, two parts of the tetracyclic ester unit, one hydroxymethyl group and two 1,4-dioxane molecules were found disordered over two positions in the asymmetric unit. Several restraints (SADI, SAME, ISOR and SIMU) were used in order to improve refinement stability.
Additionally, for compounds (+)-3d and (+)-10b one badly disordered pentane molecule was found in the asymmetrical unit and could not be satisfactorily refined.
The program SQUEEZE [7] was therefore used to remove mathematically the effect of the solvent. The quoted formula and derived parameters are not included the squeezed solvent molecule.
X-ray crystal structure analysis of (+)-3d: A colorless plate-like specimen of C34H30N2O9, approximate dimensions 0.044 mm x 0.229 mm x 0.401 mm, was used for the X-ray crystallographic analysis. The X-ray intensity data were measured. A total of 1201 frames were collected. The total exposure time was 19.15 hours. The frames were integrated with the Bruker SAINT software package using a wide-frame algorithm. The integration of the data using an orthorhombic unit cell yielded a total of 29734 reflections to a maximum θ angle of 66.59° (0.84 Å resolution), of which 5592 were independent (average redundancy 5.317, completeness = 99.6%, Rint = 9.51%, Rsig = 6.93%) and 4473 (79.99%) were greater than 2σ (F 2 (000), 808 e -. The hydrogen at N1 and N5 atoms were refined freely, but with fixed U-value und DFIX restraints.